The major proteins of the anion-exchange fractions with phospholipases A (PLA) activity were N-terminally sequenced. This chapter analyzes the bacterial membrane lipid composition of the Legionella pneumophilaaas mutants and the 130b wild type by thin-layer chro-matography (TLC). The role of Aas during L. pneumophila intracellular infection was assessed in the three host models, U937 macrophages, A549 epithelial cells, and Acanthamoeba castellanii amoebae, and Aas was found to be dispensable, because L. pneumophilaaas mutants showed the same increase in CFU in all three hosts during 72 h of infection as the wild type. The chapter presents data that show that L. pneumophila Aas contributes to the incorporation of phospholipids into the bacterial membrane, and in addition to L. pneumophila PlaA, is another enzyme involved in the detoxification of lysophospholipids. The authors examined the corresponding L. pneumophila 130b unk1 mutants for hydrolysis of diacylphospho-lipids, monoacylphospholipids, and 1-MPG and found that Unk1 did not contribute to the secreted PLA and L. pneumophila phospholipases A (LPLA) activities of L. pneumophila. The data indicate an essential role for Unk1 in infections of amoebae and macrophages by L. pneumophila. The authors identified three proteins, Aas, LvrE, and Unk1, present in the L. pneumophila culture supernatant. Aas contributes to the cell membrane integrity of L. pneumophila, but is dispensable for the infection of host cells. Since bacterial lipolytic enzymes are important bacterial tools for surviving both inside and outside of hosts, their characterization promotes one's understanding of the life cycle of L. pneumophila.

Comparative TLC analysis of cell lipids from L. pneumophila 130b wild type and aas mutants and the effect of cytolytic MPLPC on bacterial viability. (A) To analyze the cell lipids, bacteria were grown to mid-logarithmic phase and lipids were directly extracted from the cell lysates and separated by TLC (number of experiments: 2). (B) To assess the effect of cytolytic MPLPC on the viability of L. pneumophila, the wild type and two independent aas mutants were grown to mid-logarithmic phase, 0.2 mM MPLPC was added, cultures were grown for another 16 h, and the CFU were determined (number of experiments: 3). aas-9a and aas-16b represent two independent L. pneumophila 130b aas mutants. DPPE, dipalmitoylphosphatidylethanolamine; DPPC, dipalmi-toylphosphatidylcholine; MPLPC, monopalmitoylphosphatidylcholine.

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FIGURE 1

Comparative TLC analysis of cell lipids from L. pneumophila 130b wild type and aas mutants and the effect of cytolytic MPLPC on bacterial viability. (A) To analyze the cell lipids, bacteria were grown to mid-logarithmic phase and lipids were directly extracted from the cell lysates and separated by TLC (number of experiments: 2). (B) To assess the effect of cytolytic MPLPC on the viability of L. pneumophila, the wild type and two independent aas mutants were grown to mid-logarithmic phase, 0.2 mM MPLPC was added, cultures were grown for another 16 h, and the CFU were determined (number of experiments: 3). aas-9a and aas-16b represent two independent L. pneumophila 130b aas mutants. DPPE, dipalmitoylphosphatidylethanolamine; DPPC, dipalmi-toylphosphatidylcholine; MPLPC, monopalmitoylphosphatidylcholine.

Intracellular infection by L. pneumophila Philadelphia-1 wild type and an lvrE mutant as well as 130b wild type and unk1 mutants. Strains Philadelphia-1 and an lvrE mutant as well as strains 130b and the unk1 mutants unk1-cl.1 and unk1-cl.2 were used to infect cultures of U937 macrophages (A, C) or monolayers of A. castellanii amoebae (B, D) at a multiplicity of infection of 1 or 0.01, respectively. At various time points postinoculation, the number of bacteria were quantified by plating aliquots on buffered charcoal yeast extract agar. Results represent the means and standard deviations of triplicate samples and are representative of three (A, B, D) or two (B) independent experiments.

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FIGURE 2

Intracellular infection by L. pneumophila Philadelphia-1 wild type and an lvrE mutant as well as 130b wild type and unk1 mutants. Strains Philadelphia-1 and an lvrE mutant as well as strains 130b and the unk1 mutants unk1-cl.1 and unk1-cl.2 were used to infect cultures of U937 macrophages (A, C) or monolayers of A. castellanii amoebae (B, D) at a multiplicity of infection of 1 or 0.01, respectively. At various time points postinoculation, the number of bacteria were quantified by plating aliquots on buffered charcoal yeast extract agar. Results represent the means and standard deviations of triplicate samples and are representative of three (A, B, D) or two (B) independent experiments.

4. FliegerA.,, B.Neumeister, and, N. P.Cianciotto.2002. Characterization of the gene en-coding the major secreted lysophospholipase A of Legionella pneumophila and its role in detoxification of lysophosphatidylcholine.Infect. Immun.70:6094—6106.

8. MolmeretM.,, D. M.Bitar,, L.Han, and, Y.Abu Kwaik.2004. Disruption of the phagoso-mal membrane and egress of Legionella pneumophila into the cytoplasm during the last stages of intracellular infection of macrophages and Acanthamoeba poly-phaga.Infect. Immun.72:4040—4051.

9. SegalG.,, J. J.Russo, and, H. A.Shuman.1999. Relationships between a new type IV secretion system and the icm/dot virulence system of Legionella pneumophila.Mol. Microbiol.34:799—809.